My laboratory leveraged knowledge of signaling, transcriptional and translational regulators that control cardiogenesis to attempt to reprogram cardiac fibroblasts toward the cardiomyocyte state. While no single transcription factor (TF) or microRNA generates cardiomyocyte-like cells, a combination of Gata4, Mef2c and Tbx5 (GMT) induced a fundamental switch in phenotype in which mouse fibroblasts developed well-organized sarcomeres and had a broad and epigenetically stable shift in gene expression. In vitro, only a small percentage fully reprogrammed to the point of contractile activity. However, viral introduction of GMT in vivo after cardiac injury resulted in a much higher rate of cardiac fibroblasts reprogramming to contractile cells and improved cardiac function in mice. Similar results with potentially improved reprogramming upon addition of other factors were reported by others, including inhibition of Tgf- signaling by the small molecule SB431542, which results in greater quality and efficiency with GMT reprogramming. While reprogramming endogenous cardiac fibroblasts into cardiomyocyte-like cells is a promising approach for cardiac regeneration, efforts to improve the degree of reprogramming are important. To this end, we initiated studies to reveal the mechanism by which GMT fundamentally alters the cell state. Genome-wide temporal transcriptome studies and chromatin immunoprecipitation (ChIP) with each reprogramming factor demonstrate early changes that progressively reprogram fibroblasts toward the cardiomyocyte state. A deep understanding of the mechanism by which reprogramming factors can induce the cardiomyocyte phenotype through broad transcriptional and epigenetic changes will help identify factors that could improve efficiency and reveal networks that need to be activated to establish and maintain cardiomyocytes. Here, we will test the hypotheses that GMT functions dynamically in a combinatorial fashion on enhancers over time to epigenetically alter the genomic landscape, and that the epigenetic shift is enhanced by the in vivo environment and by SB431542 in vitro. We will test these hypotheses in three aims: (1) To determine the transcriptional consequences of expressing GMT during the progressive stages of in vitro and in vivo cardiac reprogramming, incorporating single cell RNA sequencing technology; (2) To determine the genome-wide occupancy of cardiac reprogramming factors during cell conversion in vitro and in vivo and to correlate them with temporal gene expression changes; (3) To define how genome-wide epigenetic changes, including histone methylation, acetylation, and DNA methylation are established during the cardiac reprogramming process and how the epigenetic landscape is altered specifically at loci occupied by reprogramming factors, resulting in transcriptional changes. Completion of these aims will reveal the complex mechanisms underlying cellular reprogramming from one somatic cell to another and will illustrate potential barriers to cardiac reprogramming that will serve as targets to improve efficiency.